A simple way of detecting or measuring particles is with the human sense of vision. However, the resolution of the human eye is limited and particles below approximately 0.05 millimetres in diameter become invisible. Microscopy instruments incorporating optical lenses can achieve a lateral resolution of about 200 nanometres. The physical properties of light limit resolution to about half the wavelength of the light used. Microscopy instruments that employ focused electrons can be used to resolve smaller particles and so extend the imaging range, further allowing for molecular-scale particles and even atoms to be detected or measured.
At the level of microbes, cells of blood, cells of other tissues or microscopic particles of organic or inorganic origin, optical instruments can be used, but the counting of such particles by microscopy has limitations and disadvantages and requires other methods. For example, the preferred method to determine microbe numbers uses a series of dilutions of the sample in question, spreading each dilution on solid microbe culture medium, incubating the culturing media and then counting the number of colonies formed by appropriately diluted samples.
The process of culturing samples amplifies single microbes from invisible cells to colonies that are detectable by the unaided human eye, but such a process has disadvantages and limitations. For example, the large numbers of vessels and materials used for culturing the microbe samples must be sterile and so must either be autoclaved for re-use, or disposed of and replaced. Other disadvantages include the time delay required to culture the microbes to sufficient numbers to be detected, the space and energy requirements to culture the microbes, and the fact that, for disease-causing pathogenic microbes, culturing of samples requires specially constructed containment facilities.
Detection and analysis of the biochemical components of life has utility in the fields of molecular biology, biochemistry, biotechnology, genetics, medicine and nanometre-scale technologies. Electron microscopy methods can be applied to the detection and analysis of molecular particles. However, electron microscopy of molecular particles suffers from the fact that high vacuum conditions are required for the microscopy method, necessitating dehydration of the sample and specialized sample preparation methods, the fact that biological particles lack sufficient contrast in the electron beam and so require a metal coating which obscures the fine detail of the particles and the fact that the high energy of the focused electron beams can shift atoms and damage surfaces.
Methods for detecting and analyzing molecular particles depend on separation methods, most typically centrifugation, electrophoresis or chromatography (See Alberts et al (1994) “Molecular Biology of the Cell”, Garland publishing, Inc., NY.). Detection methods most typically must also be applied to populations of molecules and require the use of sophisticated high-resolution optical instruments.
Many technological processes benefit from detecting and analyzing molecular particles. For example, the determination of the nucleotide sequence of nucleic acid macromolecules is most typically achieved on short (100-800) nucleotide fragments by generating families of related molecule fragments that differ in length by single nucleotides. Typically, these families of molecular fragments are separated, by electrophoresis or chromatography, and detected and analyzed to reveal the nucleic acid macromolecule sequence structure. Another example is the sequence-specific cutting of nucleic acid macromolecules by restriction endonuclease enzymes to generate a population of fragmented macromolecules which are then separated into discrete bands by electrophoresis or chromatography and detected and analyzed to reveal the sizes of the nucleic acid fragments, and so the relative positions of the restriction endonuclease cut sites. Similarly, specifically cut fragments of nucleic acid can be spliced together by a process called ligation using the enzyme ligase and the process verified by detection and analysis of the ligated fragments using electrophoresis or chromatography.
Another example is the separation and detection of populations of nucleic acid molecules generated by enzymic amplification means, such as the polymerase chain reaction (PCR) disclosed in U.S. Pat. No. 4,683,202, which allows specific nucleic acid sequences to be amplified for the purposes of detection of specific nucleic acid sequences from minute traces of starting materials.
However there are disadvantages and limitations to analyzing populations of molecular particles by electrophoresis and chromatography, in that significant amounts of molecular materials must be available to be resolved by low-resolution detection methods, the sieving materials for separation methods are expensive and the process of separation is time consuming. There is a need for rapid detection and analysis of molecular-scale particles.
Single particle detection and analysis has been achieved using scanning probe microscopes (SPMs). SPMs include the scanning tunnelling microscope (STM, See Binnig et al., “Surface studies by scanning tunnelling microscopy” Phys. Rev. Lett, 40, 57-61, 1982) disclosed in U.S. Pat. No. 4,343,993 and the atomic force microscope (AFM) of U.S. Pat. No. 4,724,318. The entire contents of these patents are incorporated herein by reference. In addition to detecting particles, SPMs are capable of manipulating and controlling particles. SPMs utilize near field probes that are operated with the probes proximal to the sample of analysis. The probe-sample separation distance is set so that the electron cloud of the probe overlaps the electron clouds of the sample and the separation distance is measured by measuring a probe-sample interaction parameter. Raster scanning of the probe over the sample allows a spatial map of the interaction parameter to be plotted to reveals ‘images’ of the surface. The resolution of the images is dependent on the geometry of the probe and atomic scale probes can be used to reveal atomic-scale detail.
In the STM electronic circuitry senses the quantum mechanical tunnel effect between the apex of a sharpened metallic probe and a conducting surface. The probe is typically fabricated from hard metal wire, such as tungsten, and has a geometry that tapers substantially from the diameter of the wire, typically 1.0-0.1 millimetres, to an apex with a radius of curvature of the order of 10 nanometres. STM is necessarily limited to imaging electrically conducting samples, or samples coated in electrically conducting materials, thereby obscuring the molecular detail of the sample.
The atomic force microscope (AFM) of U.S. Pat. No. 4,724,318 is another type of SPM that overcomes the limitations of analyzing electrically conducting samples. Atomic scale perturbations are detected between the probe and the sample by the mechanical deflection of a raster-scanned microscopic probe attached to a cantilever. The image contrast mechanism in an AFM relies on direct physical contact between the microscopic probe and the sample and so does not require a conducting sample or probe. While modern AFMs detect deflection of a cantilever by measuring the deflection of a laser beam reflected from the cantilever surface, the AFM of U.S. Pat. No. 4,724,318 utilizes a tunnel tip attached to a z-drive in the form of a piezoelectric element.
The STM has imaged single nucleic acid macromolecules (see Guckenberger et al., “Scanning tunneling microscopy of insulators and biological specimens based on lateral conductivity of ultrathin water films” Science, 266, 1538-1540, 1991) and also sub-molecular components of nucleic acids, the purine (See Heckl et al “Two-dimensional ordering of the purine base guanine observed by scanning tunneling microscopy” PNAS, 88, 8003-8005, 1991) and pyrimidine bases (See Sowerby et al., “Scanning tunneling microscopy of uracil monolayers self-assembled at the solid/liquid interface” J. Electroanal. Chem, 433, 85-90, 1997). A method of STM imaging for single molecule nucleic acid sequencing has been disclosed (See Heckl et al., “DNA base sequencing” Nonlinear Optics, 1, 53-59, 1992) and U.S. Pat. No. 5,106,729, U.S. Pat. No. 5,270,214 and U.S. Pat. No. 5,620,854, the entire contents of which are incorporated herein by reference. However, molecular characterization by SPM is dependent on molecular resolution detection means by the SPM and suffer from the disadvantage that SPM probes are substantially short-lived and that the preferred graphite substrate used for adsorbing nucleic acid is substantially rich in artifacts which mimic DNA structure (See Clemmer et al., “Graphite: a mimic for DNA and other biomolecules in scanning tunnelling microscopy studies” Science, 251, 640-642, 1991).
Another type of apparatus that is available for particle analysis is the Coulter counter, as disclosed in U.S. Pat. No. 2,656,508, comprising two substantially isolated reservoirs of electrically conductive ionic fluid separated from each other by a substantially, electrically insulating barrier containing a small aperture that is the only conduit through which particles can pass between the reservoirs. Electrodes placed in each reservoir provide a means to generate a current of ions through the aperture by the application of a potential difference across the electrodes.
The effective cross-sectional area of the aperture limits the flux of ions traversing the aperture, and the length of the aperture is typically between 70 and 100 percent of the aperture diameter. Particle sensing is achieved with this configuration by utilizing a general principle known as resistive pulse sensing (See Bayley et al., “Resistive Pulse Sensing—From Microbes to Molecules” Chem. Rev., 100, 2575-2594, 2000). According to this principle, the transit of a particle suspended in the ionic fluid passing through the aperture causes a resistive pulse signal in the electrical conductivity of the aperture as the particle displaces ions within the aperture, so reducing the measured current density for the period of time that the particle occupies the aperture. Studies have shown that the magnitude of the resistive pulse signal is proportional to the volume occupied by the particle within the aperture. The lower size limit for detection in these devices is reached when the particle size generates a resistive pulse signal that cannot be distinguished from the background noise of the ionic current through the aperture. Coulter-type devices are limited to detecting particles in the range of 2 percent to 60 percent of the aperture diameter, which necessitates that apertures must be fabricated for specific particle sizes. Coulter-type devices have found commercial utility analyzing particles in the range ˜0.4 micrometres to ˜1.0 millimetres utilizing apertures ranging in size from 20 micrometres to 2 millimetres.
U.S. Pat. No. 4,853,618 discloses a Coulter-type particle analysis apparatus in which an aperture is varied by the precisely controlled automatic insertion of an insert into the aperture so as to reduce the effective cross-sectional area of the aperture.
Coulter-type analysis of molecular scale particles has been achieved using molecular-scale apertures of biological origin. U.S. Pat. No. 5,795,782 and U.S. Pat. No. 6,015,714 disclose a method utilizing the α-hemolysin aperture for rapid nucleic acid sequence determination and molecular characterization. Despite promising laboratory evidence, apertures based on protein pores suffer numerous disadvantages and limitations in that their formation relies on stochastic self-organization processes so that they are unpredictable and difficult to fabricate and the proteins from which they are constructed and the biological membranes into which they are inserted have short functional lifetimes.
Solid-state nanometre-scale apertures 1.5 nanometres in diameter have been fabricated in a silicon nitride membrane by focused ion beam lithography and have demonstrable DNA sensing utility (See Li et al., “Ion-beam Sculpting at Nanometre Length Scales” Nature, 412 166-169, 2001).
In U.S. Pat. No. 6,413,792, solid-state nanometre-scale apertures have been disclosed where utility is claimed for ultra-fast nucleic acid sequencing methods. U.S. Pat. No. 6,706,203 and US 2003/0080042 disclose an adjustable nanopore, nanotome and nanotweezer comprising two sliding solid-state crystalline or ceramic window apertures overlaid to create a single smaller aperture. However such solid-state apertures are difficult to construct and is constrained to a small window size.